Download Mycobacterium Strain and Type of Resistance in Pulmonary

Shiraz E-Med J. 2015 July; 16(7): e27748.
DOI: 10.17795/semj27748
Research Article
Published online 2015 July 22.
Mycobacterium Strain and Type of Resistance in Pulmonary Tuberculosis
Patients: A Missed Link in Iran’s National Tuberculosis Plan
1,*
2
3
4
Behnam Honarvar ; Mohsen Moghadami ; Amir Emami ; Abbas Behzad Behbahani ;
5
6
5
7
Mohammad Taheri ; Amir Roudgari ; Golnar Sami Kashkoli ; Mohsen Rezaee ; Ehsan
6
5
6
7
Farzanfar ; Zahra Zaree ; Jamalodin Goharnejad ; Fatemeh Khavandegaran ; Kamran
1
Bagheri Lankarani
1Health Policy Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
2HIV/AIDS Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
3Bactriology and Virology Department, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz, IR Iran
4Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, IR Iran
5Central Microbiology Laboratory, Shiraz Health Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
6Shiraz University of Medical Sciences, Shiraz, IR Iran
7Shiraz Health Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
*Corresponding Author: Behnam Honarvar, Health Policy Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran. Tel/Fax: +98-7132309615, E-mail: [email protected]
Received: February 6, 2015; Revised: April 12, 2015; Accepted: July 11, 2015
Background: The Incidence of multi-drug resistant tuberculosis (MDR-TB) is constantly increasing.
Objectives: This study aimed to clarify an important missed link in Iran’s national TB plan.
Patients and Methods: Through a 9-month period, all pulmonary TB patients diagnosed based on the national TB protocol, in Shiraz TB
center, were selected and culture of TB colonies, drug susceptibility testing (DST), polymerase chain reaction (PCR) for detection of IS6110
gene, Isoniazid (INH) and Rifampin (RIF) tuberculosis resistance were done to collect data. Data were analyzed using SPSS.
Results: In 92 included patients, (mean age 45.4 ± 15 years), DST results showed that 16 cases (17.4%) were resistant to INH, 19 (20.7 %) to RIF
and 24 (26.1%) to both INH and RIF. Polymerase chain reaction identified IS6110 gene in 71 cases (77.2%) and gene mutations in 3 (3.2%) KatG,
3 (3. 2%) InhA, 9 (9.7%) both KatG and InhA, 17 (18.4%) rpoB and 20 (21.7%) in KatG, InhA and rpoB genes. Patients with INH-resistant tuberculosis
were more than those with RIF-resistant (OR = 7.1).
Conclusions: Findings of the present study show that 4 out of five new cases of pulmonary TB patients who were diagnosed based on the
national TB protocol (clinical symptoms and acid fast bacilli staining) had IS6110 gene (MTB, Mycobactrium TB) and at least one-fifth of this
group had A kind of Drug Resistant TB. Therefore, by using PCR ,as a complementary test, it could be possible to start 1st line anti-TB drugs
for only MTB cases (up to 77% of the patients) and 2nd line drugs for MDR cases (15% of cases). This policy aims to achieve safety and better
outcome for patients while saving human and financial resources in health care system.
Keywords: Tuberculosis; Drug Resistant; Gene; PCR
1. Background
Despite political, economic, research, and community
efforts, Tuberculosis (TB) remains one of the world’s deadliest communicable diseases (1). In 2013, an estimated 9.0
million people developed TB and 1.5 million died from
the disease (2). Also, an incidence rate of TB reported in
Iran is 21 cases per 100,000 people (3).
An important challenge in TB control is the emergence
of resistant strains to the most potent anti-TB drugs (4).
Multidrug Resistant Tuberculosis (MDR-TB), is caused by
mycobactria isolates which are resistant to, at least, Isoniazid (INH) and, Rifampin (RIF) (5, 6). Delayed diagnosis
and inappropriate treatment of tuberculosis may result
in constantly increasing severity, mortality, spreading
and the emergence of MDR-TB (7-9). Annually, MDR-TB is
estimated to afflict 490,000 cases, or 5% of the global TB
burden. This appears to be a big challenge to TB control
due to its complex diagnostic and treatment problem
(10, 11).
On the other hand, successful control of TB and achievement of third Millennium Development Goals (MDGs)
for elimination of TB by 2050 depends on the extent of
timely diagnosis of patients with TB and their successful
treatment (12). Therefore, early and rapid detection of
multidrug resistance is a global priority and is essential
for efficient treatment and control of Mycobacterium TB
(MTB) (13). IS6110 is an insertion sequence specific to Mycobactrium TB which can be used to differentiate MTB
species from other mycobacteria (14).
Copyright © 2015, Shiraz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
Honarvar B et al.
Isoniazid resistance is mostly associated with increased
risk of treatment failures and acquiring new drug resistance (15, 16). Isoniazid enters the bacterial cell wall as a
prodrug and is converted to a toxic substance in the cell
by a catalase peroxides encoded by a KatG gene. Any mutation in this gene can confer bacterial resistance to isoniazid (17, 18).
Also, InhA is a part of a very long chain fatty acid, mycolic acids, in elongation system whose products represent catalysis in the last step of fatty acid elongation. Any
mutation in InhA sequences makes changes in INH target
and makes the bacteria resistant to isoniazid (19, 20).
Rifampin, as an anti TB drug, was introduced in 1972 and
showed excellent sterilizing activity. It acts by binding to
the β-subunit of RNA polymerase (rpoB) (5), the responsible enzyme for transcription and expression of mycobacterial genes, inhibits the bacterial transcription activity
and results in killing the organism. Any mutation in the
81-bp core region of rpoB was reported to be responsible
for drug resistance in at least 95% of the isolates (21, 22).
Using molecular methods for identification of mutations in the genes may offer means for rapid screening
of the drug resistance among the MTB isolates and initiation of early treatment (23, 24).
2. Objectives
The aim of this study was to determine the prevalence
of MTB among patients who were diagnosed based on the
national TB program (clinical symptoms and Acid Fast Bacilli sputum smear staining) and were treated routinely
as pulmonary TB.
Also, this study aimed to detect different patterns of
drug resistance, including MDR-TB among these patients
to show the ratio of patients that should not be treated as
TB and ratio of patients that must be treated as MDR-TB
from the onset of diagnosis. Risk factors associated with
resistant patterns were also examined in this study.
3. Patients and Methods
3.1. Setting
This molecular study comprised of 92 new cases (from
December 2012 to August 2013) who were diagnosed
based on clinical symptoms and Acid Fast Bacilli (AFB)
staining and planned to be treated as pulmonary TB at TB
referral centers affiliated to Shiraz University of Medical
Sciences, south of Iran. Demographic data for each patient was obtained from the national TB-registry system.
3.2. Acid Fast Bacilli Staining and Culture on Solid
Media
Concentrated sediment of sputum samples were collected from all patients as follows: Sputum samples
were liquefied and decontaminated with N-acetyl cysteine-2.5%, Sodium Hydroxide (NAOH) and concentrated by
2
centrifugation at 3000 rpm for 20 minutes (10). The sediment was used to inoculate onto a Lowenstein-Jensen (LJ)
medium. Inoculated media were incubated at 37ºC for 8
weeks and examined weekly for colony formation.
3.3. Niacin Accumulation and Nitrate Reduction Test
Niacin test was carried out on suspected buff colonies
cultured for 6 weeks. Then Niacin positive colonies were
used for nitrate reduction test (25).
3.4. Drug Susceptibility Testing
For this purpose, colonies of MTB were taken from the
surface of the LJ slant. Drug susceptibility testing (DST) was
performed using INH (0.2 mg/L. Sigma-Aldrich) and RIF (40
mg/L. Sigma-Aldrich) according to the proportion method
of Canetti (12). For each set of DST, a known MDR strain was
used as positive control and H37Rv strain provided by Tehran mycobacteriology research center as negative control.
3.5. DNA Preparation
The DNA was extracted from colonies with QIAamp DNA
mini kit (QIAgen, Inc., Valencia, California, and USA) according to the manufacturer’s instruction. The extracted
DNAs were stored at -70ºC.
3.6. IS6110 Detection
Polymerase chain reaction (PCR) was performed on extracted DNAs using specific primers TB1 (5/-ATC CTG CGA
GCG TAG GCG TCG G-3/) and TB2 (5/-CAG GAC CAC GAT CGC
TGA TCC GG-3/) for a 190 bp fragment. Polymerase chain
reaction was performed in a total volume of 50µL reaction
mix of 5 µL PCR buffer (10x-Qiagene Inc.), 1.5 µL MgCl2 (50
mM-Qiagene Inc.), 1 µL Deoxynucleotide triphosphates
(dNTPs) (0.2 mM-Qiagen, Inc.), 1.5 U Taq DNA polymerase
(Qiagen, Inc.) and 1 µL of each primer (10 pmol/µL) with 5 µL
of template. Nuclease free sterile double distilled water was
added to a final volume of 50 μL. The mixture was amplified
using thermo cycler program including 10 minutes at 95ºC
for initial denaturation, followed by the PCR condition at
94ºC for 45 second, 65ºC for 1 min and 72ºC for 45 seconds,
for 35 cycles with final extension at 72ºC for 10 minutes. The
PCR products were run on 2% agarose gel electrophoresis,
and visualized after staining with ethidium bromide.
3.7. Molecular Detection of Rifampin Resistance
In this study, a multiplex PCR was performed to detect mutation in rpoB region in all positive IS6110 gene
samples using the following sets of primers: rpoB516
(5/-CAGCTGAGCCAATTCATGGA-3/), rpoB526 (5/-CTGTCGGGGTTGACCCA-3/), rpoB531(5/ CACAAGCGCCGACTGTC-3/) and RIRm (5-TTGACCCGCGCGTACAC-3) for 218bp,
185 bp, and 170 bp fragments, respectively. Polymerase
chain reaction was done in a total volume of 25 µL reaction mix containing 2.5 µL PCR buffer (10x-Qiagene
Inc.), 4 µL MgCl2 (5.5 mM-Qiagene Inc.), 1 µL dNTPs (0.2
Shiraz E-Med J. 2015;16(7):e27748
Honarvar B et al.
mM-Qiagen, Inc.), 1.5 U Taq DNA polymerase (Qiagen,
Inc.) and 1 µL of each primer (10 pmol/µL) with 5 µL of
template. Nuclease free sterile double distilled water
was added to a final volume of 25 μL. The mixture was
amplified with the following thermo cycler program:
5 minutes at 95ºC for initial denaturation, followed by
the PCR condition at 95ºC for 30 seconds, 68ºC for 30
seconds and 72ºC for 30 seconds, for 40 cycles with the
10 minutes final extension at 72ºC. The PCR products
were examined for banding patterns by 8% poly acrylamide gel electrophoresis, and visualized by ethidium
bromide staining. The absence of each fragment in the
electrophoresis pattern represented mutation in that
region and was reported as Rifampin resistance (RIFr).
3.8. Molecular Detection of Isoniazid Resistance
In this study, KatG and InhA were considered as target
genes for detecting INH resistance using specific sets of
primer for PCR with the following specifications: KatGOF
(5/-GCA GAT GGG GCT GAT CTA CG-3/), KatG5R (5/-ATA CGA
CCT CGA TGC CGC-3/) and InhAP-15 (5/-GCG CGG TCA GTT
CCA CA-3/), InhAPF2 (5/-CAC CCC GAC AAC CTA TCG-3/) for a
292 bp and 270 bp fragments, respectively. To detect these
two types of gene mutations, PCR was performed as described for RIFr. The absence of any fragment in the electrophoresis gel was regarded as mutation in that region
and reported as INHr.
3.9. Statistical Analysis
All data were analyzed by SPSS software version 11.5 (SPSS,
Chicago, Illinois, USA). The accuracy of data was ensured
by randomly selecting and checking completed questionnaires against their corresponding data in the SPSS software. Chi-squared, Fisher’s exact, and t-tests were the appropriate tests used in this study. After univariate analysis,
correlation of the independent variables with P ≤ 0.2 and
resistance to anti-TB drugs (as dependent variables) were
assessed by binary logistic regression (forward model). P
values less than 0.05 were considered significant.
3.10. Ethics Statement
The protocol of this study was approved by Ethics Committee of the Health Policy Research Center affiliated to
Shiraz university of medical sciences. All patients’ data
were kept confidential and all drug-resistant patients
were treated and cared for accordingly.
4. Results
This study was comprised of 92 patients whose mean age
was 45.4 ± 15 years with male to female ratio of 1.9 (Table 1).
Twenty three subjects (25%) had history of imprisonment
and 14 (15.2%) were intravenous drug users (IDUs). Nine
(9.8%) were HIV-positive and 8 (8.7%) had diabetes mellitus
(DM). Three (3.3%) had a history of prolonged corticosteroid
treatment and were on prolonged treatment course of corticosteroids and 4 (4.3%) had cancer. In the past history, 87
Shiraz E-Med J. 2015;16(7):e27748
(94.6%) had cough for more than two weeks, 80 (87%) had
weight loss and 30 (33%) exhibited hemoptysis.
Direct smear AFBs was positive in 65 cases (70.7%) and
negative in 27 (29.3%). Culture was positive in 60 cases
(66.7%), including 57 direct smear AFBs positive and 3
direct smear AFBs negative. Forty six (50%) had Niacin
and Nitrate reduction tests positive. Seventy one (77.2%),
including 53 culture positive and 18 culture negative
showed IS6110 gene in their PCR (Figure 1).
Table 1. Demographic and Risk Factors of Drug Resistant Suspected Tuberculosis Patients (n = 92) Referred to Tertiary Level
TB Centers Affiliated With Shiraz University of Medical Sciences,
South of Iran a
Variable
Demographic items
Age, y
Median
Minimum
Maximum
Gender
Male
Female
Nationality
Iran
Afghan
Marital status
Single
Married
Place of living
Urban
Rural
Job
Employed
Unemployed
Risk Factors
History of imprisonment
Yes
No
Prior TB or receiving Anti-TB treatment
Yes
No
Intravenous drug injection (IDUs)
Yes
No
HIV infection
Yes
No
Diabetes mellitus
Yes
No
Being on corticosteroid
Yes
No
a Data are presented as No. (%) or Mean ± SD.
Values
45.47 ± 15.05
49
11
80
64 (69.6)
28 (30.4)
75 (81.5)
17 (18.5)
26 (28.3)
66 (71.7)
64 (69.6)
28 (30.4)
43 (46.7)
49 (53.3)
23 (25)
69 (75)
27 (29.3)
65 (70.7%)
14 (15.2)
78 (84.8)
9 (9.8)
83 (90.2)
8 (8.7)
84 (91.3)
3 (3.3)
89 (96.7)
3
Honarvar B et al.
Figure 1. PCR Amplification of 190 bp on Agarose Gel Electrophoresis for
IS6110 Detection
Figure 3. Polymerase Chain Reaction Amplification of 218 bp, 185 bp and
170 bp on 8% Poly Acryl Amide Gel Electrophoresis for rpoB (516, 526, 531)
in Mycobactrium TB Respectively
Lane 1: 100 bp ladder, Lane 2 to 8 patient samples, PC: Positive Control, NC:
Negative Control.
Figure 2. Polymerase Chain Reaction Amplification of 292 bp and 270
bp on Agarose Gel Electrophoresis for KatG and InhA in Mycobactrium TB
Respectively
Lane 1: 100 bp ladder, Lane 2 to 8 patient samples, PC: Positive Control, NC:
Negative Control.
Table 2. Pattern of Drug Resistance and Gene Mutation in Drug
Resistant Suspected Tuberculosis Patients (n = 92) Referred
to Tertiary Level TB Center Affiliated With Shiraz University of
Medical Sciences, South of Iran a,b
Test Method
Test Target
Frequency
DST
16 (17.4)
Resistant to Isoniazid
Culture
PCR
KatG
inhA
Lane 1: 100 bp ladder, Lane 2 to 7 patient samples, PC: positive Control, NC:
Negative Control.
Sixteen cases (17.4%) were resistant to INH, 19 (20.7 %) to RIF
and 24 (26.1%) to both INH and RIF according to DST (Table
2). The PCR amplification showed that 3 cases (3.3%) had
KatG and 3 (3.3%) had InhA gene mutations (Table 2, Figure 2)
and 9 (9.8%) revealed both KatG and InhA mutation (Table 2).
Moreover, PCR detected rpoB gene mutation in 17 cases
(18.5%) (Table 2, Figure 3). Twenty subjects (21.7%) harbored
KatG, InhA and rpoB gene mutations (Table 2). Seventy-eight
(85.7%) patients showed positive findings compatible with
TB in their chest X-ray. Considering different variables and
various patterns of drug resistance, only patients who were
resistant to INH showed a significant correlation with resistant to RIF (OR = 7.1, CI (95%) = 1.9 - 26.8, P = 0.004).
4
KatG/inhA
Resistant to Rifampin
Culture
PCR
DST
repo B
Resistant to both
Isoniazid and Rifampin
Culture
PCR
DST
KatG/inhA/repo B
3 (3.3)
3 (3.3)
9 (9.8)
19 (20.7)
17 (18.5)
24 (26.1)
20 (21.7)
a Abbreviations: DST, drug susceptibility testing; PCR, polymerase
chain reaction.
b Data are presented as No. (%).
Shiraz E-Med J. 2015;16(7):e27748
Honarvar B et al.
5. Discussion
Tuberculosis remains one of the most challenging issues in global health. An important challenge for TB control is the spread of strains that are resistant to the most
potent anti-TB drugs (26) and have been reached an emergent and epidemic proportion in many countries (27-29).
This specifically applies to MDR-TB and it’s relation to
poor treatment outcomes and high rates of case-fatality
(30). Approximately, 3.7% of recent and 20% of previously
TB treated cases are afflicted with MDR-TB (31). However,
less than 5% of the existing MDR-TB patients are currently
being diagnosed as a result of serious laboratory capacity
constraints which results in delayed MDR-TB diagnosis
causes, prolonged treatments and ever-increasing costs
(32). Therefore, the early and rapid detection of multidrug resistance using molecular techniques have the
potential to significantly hasten the diagnosis and initiation of appropriate treatment (33).
Results of this study showed that no more than 80% of
new cases of pulmonary TB patients that were diagnosed
based on national TB protocol (clinical symptoms and
AFB sputum smear staining) had MTB (IS6110 gene) and
at least one-fifth of this group had MDR-TB. These results
show that at least 1 out of every 5 patients who was routinely diagnosed and treated as a new case of pulmonary
TB in our region, lacked the IS6110 gene. This finding is
more than the result found by another study in Thailand
which showed the lack of IS6110 gene in 10% of patients
(34) and less than 31% of the cases in India who had low
to zero copy number of IS6110 gene (35) and much less
than 85% of MDR-isolates in Tehran that did not have this
gene (36). Therefore, the possibility of less frequent genes
markers for MTB, infection by atypical mycobacteria or
other diagnosis should be kept in mind in negative IS6110
gene patients (37, 38).
According to DST in our study, 17.4%, 20.7% and 26.1% of
IS6110 gene positive patients were resistant to INH, RIF
and both drugs, respectively. Another study in tertiary
level TB center in Iran revealed that 2.6%, 0.9% and 6.3% of
new cases compared to 3.6%, 3.2% and 31.7% of previously
treated TB patients had INH, RIF and MDR-TB, respectively
(39). In 2003 - 2004, it was reported that 2.6% of new cases
and 56% of previously treated TB patients that referred to
the same center in Tehran had MDR-TB (40). In Saudi Arabia INH, RIF resistant cases composed of 33.8%, and 23.5%
of cases were based on DST respectively besides the presence of MDR in 20.6% of patients (41). The discrepancy of
these results could be explained in terms of different settings of studies regarding population, method, location
or time of study.
In this study, 24 (68%) and 20 (62%) cases were proved to
be resistant to both INH and RIF by DST and molecular assay, respectively. This was in concordance with the result
of another study in Philippines that found MDR as the
most frequent resistance pattern among TB patients (42).
We found that 3 (3.2% of all patients, 9.3% of MDR patients
Shiraz E-Med J. 2015;16(7):e27748
and 20% of INH resistant cases) had KatG or InhA gene mutation and 9 (9.7% of all patients, 28% of MDR patients and
60% of INH resistant cases) exhibited mutation in both
genes by Allele Specific PCR in this study.
Another study in Tunisia (43) detected that 96.4% and
3.6% of INH-resistant isolates had KatG and InhA gene mutations, respectively. In that study, 66% of RIF-resistant isolates yielded the rpoB gene mutation, that was less than
all RIF-resistant isolates in our study that were affected
by this kind of gene mutation. In a study carried out in
Northwest of Iran (44), it was concluded that 76% of INHresistant strains showed KatG gene mutation, which was
much higher than 20% found in our study. Another molecular study performed in Egypt demonstrated that 92.3%
and 86.9% of INH and RIF resistant cases were caused by
KatG and rpoB genes mutation, respectively (45). In a study
in Sudan, it was claimed that 12% (vs 3/71; 4% in our study)
and 8% (vs 17/71; 23.9% in our study) of MTB isolates had KatG
and rpoB genes mutation, respectively (45). In Ethiopia, 35
out of 260 cases (13.4%) of smear positive pulmonary TB
showed INH resistant resulting from KatG gene mutations
in 33 cases (12.7%) and InhA gene mutations in 2 cases (0.7%)
(46). In that study, 12 cases (4.6%) had rpoB gene mutation
and 13 (5%) were MDR-TB, compared to respective frequencies of 18.4% and 21.7% in our study (46).
Among different patterns of drug resistant and factors
that were assessed, only those resistant to INH showed a
significant correlation with resistant to RIF (OR = 7.1). A
study in Tehran, Iran proved that anti-TB drug resistance
had more correlation with age under 45 years, male sex,
previous TB treatment, immigration, poor living conditions, and unemployment (39). In another study it was
concluded that age > 65 years was associated with higher possibility of MDR-TB (40). In Bangladesh, younger
age, peri-urban locality, history of contact and tuberculosis in the past and socioeconomic status were associated with a higher rate of MDR-TB (47). A study from China showed that, inappropriate treatment, retreating,
age, financial burden, poor knowledge, side effects of
TB treatment and lack of service coordination were the
influencing factors in the development of MDR-TB (17).
A case control study in China, showed that more than
three TB foci in the lung, nonstandard or irregular therapy, and adverse effects of anti-TB drugs, were associated
with MDR-TB in previously treated TB patients (48). In a
systematic review, it was found that MDR-TB cases in Europe were more foreign borne [odds ratio (OR) 2.46; 95%
CI 1.86 to 3.24], younger than 65 years (OR 2.53; 95% CI
1.74 to 4.83), males (OR 1.38; 95% CI 1.16 to 1.65), and HIV
positives (OR 3.52; 95% CI 2.48 to 5.01) (49). Ohmori et al
concluded that TB patients in Japan who are under 80,
foreigners and retreated cases are more susceptible to
TB drug resistance and especially MDR-TB (50). In California, previous anti-TB treatment was associated with
MDR-TB (OR 6.57) (51). A review study revealed that previ5
Honarvar B et al.
ous TB treatment and duration of treatment, immigration, alcoholism and HIV co-infection were risk factors
for developing extensively drug resistant-TB (XDR-TB)
(52). We found that 18.7% and 15.7% of INH and RIF resistant cases and 16.6% of MDR-TB patients were Afghan
nationals, which were considerably less than 66.5% of
MDR-TB patients that had the same nationality in other
tertiary level TB center study in Iran (39).
The limitation of this study was that we used sets of
primers that in the mutated forms could not be annealed
and amplified with specific sequences, in contrast to the
studies that mutation evaluation was based on sequencing methods.
Considering the increasing rate of MDR-TB, putting
patients on anti-TB drugs treatment courses based on
diagnosis provided only by clinical symptoms and AFB
staining may cause overtreatment in at least 20% and inappropriate treatment in about 15% of patients who are
suspected to have pulmonary TB. Therefore, molecular
studies as a complementary diagnostic tool help decrease
mentioned pitfalls and as a result would help to achieve
patients’ safety and their better outcome while saving
human and financial resources in health care systems.
We suggest strengthening the referral TB laboratories in
Iran with molecular studies’ needed infrastructures and
facilities.
Acknowledgements
The researchers gratefully acknowledge the contribution of the patients referred to the TB centers affiliated to
Shiraz University of Medical Sciences, all personnel of TB
center for their valuable cooperation during the course
of this study and department of research affiliated to Fars
provincial government for financial support of this work.
We also thank Sara Sedighi for improving the use of English in this manuscript.
Funding/Support
This study was funded and supported by the department of research affiliated to Fars provincial government.
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